Genetic Engineering and Its Applications

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Chapter 17

• Genetic Engineering and Its Applications

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Chapter 17
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Genetic Engineering

• Key discoveries that made genetic engineering
  possible
• The ability to cut DNA at specific sites and join
  different fragments together.
• The ability to transform different organisms with
  foreign recombinant DNA.

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Genetic Engineering

• Genetic engineering is being applied to medicine,
  industry, and agriculture.

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A Case Study Manufacturing Proteins from
Recombinant DNA

• Pituitary dwarfism is due to a single gene
  mutation.
• Type I dwarfism is a recessive trait due to lack
  of GH1 growth hormone protein.
• Treatment consisted of injections of GH1
  extracted from cadaver pituitaries.
• A few cases of Creutzfeldt-Jakob syndrome
  appeared in patients who received pituitary GH1,
  indicating the cadaver supply was contaminated
  with prions.

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A Case Study Manufacturing Proteins from
Recombinant DNA

• Pituitary dwarfism is due to a single gene
  mutation.
• To develop a safer source of GH1, the GH1 gene
  was isolated and cloned into a plasmid using
  restriction enzymes. (Fig. 17.1)
• Bacterial cells were transformed with the
  recombinant plasmid.
• A probe for GH1 confirmed that selected bacterial
  cells contained the gene. (Fig. 17.2)
• Large quantities of GH1 protein are now produced
  by growing GH1-expressing bacterial cells.

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Figure 17.1, upper
1. Isolate mRNAs from cells in pituitary gland.
2. Use reverse transcriptase to synthesize a
cDNA complement to each mRNA. Use DNA polymerase
to make each cDNA double-stranded.
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Figure 17.1 middle
3. Attach a restriction enzyme recognition site
to ends of each cDNA.
4. Cut cDNAs and plasmids with restriction
enzyme remaining sticky ends anneal by
complementary base pairing.
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Figure 17.1 lower
5. Ligate cDNAs and plasmids with DNA ligase.
6. Introduce recombinant plasmids into E. coli
cells to create cDNA library.
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Figure 17.2 upper
FINDING THE GROWTH HORMONE GENE IN A cDNA LIBRARY
1. Grow E. coli cells containing plasmids on many
plates. Each colony contains a different cDNA.
2. Lay a filter on each plate, then remove. Some
cells from each colony stick to filters.
3. Treat filters with chemical to make DNAs
single stranded.
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Figure 17.2 lower
FINDING THE GROWTH HORMONE GENE IN A cDNA LIBRARY
Labeled probe
4. Probe filters with labeled DNA (short sequence
inferred from amino acid sequence of growth
hormone).
5. Probe binds to growth hormone gene. Lay X-ray
film over filters black spot marks location of
probe.
E. coli containing growth hormone gene
6. On original plates, find colony of E. coli
cells that contains growth hormone gene. Sample
cells, grow, and analyze.
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A Case Study Manufacturing Proteins from
Recombinant DNA

• Ethical concerns should physicians be allowed
  to prescribe GH1 indiscriminately to individuals
  who are short, or are athletes, but who have
  normal levels of GH1?

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A Case Study Using Pedigree Analysis to Find a
Gene

• Huntingtons disease is an inherited genetic
  disorder that develops later in life and is
  fatal.

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A Case Study Using Pedigree Analysis to Find a
Gene

• Pedigree analysis reveals patterns of inheritance
  that can be used to help find a gene, if a trait
  is due to a mutation in a single gene.
• Genes located on sex chromosomes the pattern of
  inheritance differs in males and females, and
  skips generations. (Fig. 17.4)
• Autosomal recessive traits the pattern of
  inheritance is the same in males and females,
  and may skip generations. (Fig. 17.5a,b)

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Figure 17.4a
Human sex chromosomes
Females XX
Males XY
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Figure 17.4b
Occurrence of hemophilia in royal families of
Europe
I
Prince Albert
Queen Victoria
II
III
IV
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Figure 17.5a
Pedigree of a family with Huntingtons disease
I
Unaffected male
Affected female
II
III
IV
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Figure 17.5b
Pedigree of a family with autosomal recessive
disease
I
Female
Male
II
III
IV
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A Case Study Using Pedigree Analysis to Find a
Gene

• Pedigree analysis reveals patterns of inheritance
  that can be used to help find a gene, if a trait
  is due to a mutation in a single gene.
• Autosomal dominant traits the pattern of
  inheritance is the same in males and females and
  does not skip generations.
• Huntingtons disease is an autosomal dominant
  trait. (Fig. 17.3)

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Figure 17.3
Unaffected male
Unaffected female
Affected male
Unaffected female
I
Each row represents a generation
II
III
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A Case Study Using Pedigree Analysis to Find a
Gene

• The precise location of the gene is determined by
  mapping and sequencing.
• The gene is mapped by finding known genetic
  markers with which it co-inherits. (Fig.
  17.6a,b)
• The Huntington allele was mapped to a region near
  a few known restriction enzyme recognition sites.
  (Fig. 17.7)
• Single nucleotide polymorphisms can now be used
  as much more precise markers.

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Figure 17.6a
Restriction enzyme cuts produce DNA fragments of
various lengths
Sites where restriction enzyme cuts DNA
Section of chromosome
Polymorphic sites some individuals have this
site, some dont
2.5 kb
15 kb
8.4 kb
3.7 kb
1.2 kb
2.3 kb
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Figure 17.6b
A
B
C
D
17.5 kb 15.0 kb
17.5 kb 15.0 kb
Both polymorphic sites present
8.4 kb
8.4 kb
Left present, right absent
4.9 kb
4.9 kb
Both polymorphic sites absent
Left absent, right present
3.7 kb
3.7 kb
2.5 kb 2.3 kb
2.5 kb 2.3 kb
1.2 kb
1.2 kb
If relatives who share a particular banding
pattern also share an inherited illness, then the
gene responsible for the disease is located near
this region of their genome.
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Figure 17.7
I
II
III
IV
AB
AA
V
AB
AB
BC
AB
AB
AB
AB
BC
AB
AB
BC
BB
BC
AC
AA
BC
CD
BC
BC
AB
AB
VI
CC
BC
BC
AA
BC
AA
BC
AA
AB
AC
AC
AC
AC
AC
VII
BC
BC
AC
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A Case Study Using Pedigree Analysis to Find a
Gene

• The precise location of the gene is determined by
  mapping and sequencing.
• The locus to which an allele maps is sequenced to
  identify variations that are present in all
  diseased individuals but absent in healthy
  individuals.
• The Huntington disease allele was found to have
  more copies of a CAG repeat than the normal
  allele.
• The CAG repeat codes for polyglutamine, which
  accumulates as aggregates in cells of the brain.

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A Case Study Using Pedigree Analysis to Find a
Gene

• Finding the gene makes new approaches to therapy
  possible.
• A genetic test can be developed.
• The molecular nature of the disease can be used
  to find drugs that may alleviate symptoms.

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A Case Study Gene Therapy as a Cure for Disease

• For successful gene therapy, the disease allele
  must identified and transferable into diseased
  individuals such that the gene is expressed in
  the right location.
• Retroviruses have been used to deliver genes to
  specific tissues, and get them incorporated into
  host DNA.
• Adenoviruses can deliver genes to the respiratory
  tract, but cannot incorporate the DNA into the
  host chromosome.

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A Case Study Gene Therapy as a Cure for Disease

• Only one case of gene therapy has led to a cure
  for a disease.
• SCID-X1 is a defect in a growth factor receptor
  protein on T cells that leads to defective T
  cells and a compromised immune system.
• The normal allele for the receptor was delivered
  to bone marrow stem cells of 2 patients via a
  retrovirus.
• The stem cells were returned to the patients'
  bone marrow and began producing normal T cells.

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Biotechnology in Agriculture

• Three strategies for altering crops have been
  used
• Introduction of the Bt toxin gene so that plants
  can make their own insecticide.
• Introduction of resistance to the herbicide
  glyphosate.
• Improvement of the nutritional quality of crops
  by changing the chemical content of seeds.
  Example golden rice (Fig. 17.10a,b)

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Figure 17.10a
GENETIC ENGINEERING OF Ti PLASMIDS
Tumor-inducing genes
1. Start with normal Ti plasmids.
T-DNA
2. Remove tumor-inducing genes.
T-DNA
Promoter
3. Add genes for enzymes required for ß-carotene
synthesis along with promoter that is activated
in endosperm.
Genes for three enzymes
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Figure 17.10b
Rice plants infected with transformed
Agrobacterium produce ß-carotene in their seeds.
Golden rice (transformed)
Control(not transformed)
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Figure 17.8
Geranyl geranyl diphosphate (GGPP)
Enzyme 1
Phytoene
Enzyme 2
Lycopene
Enzyme 3
ß-carotene
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Biotechnology in Agriculture

• Controversies over genetically modified foods
  exist.
• Proponents point to reduced usage of pesticides
  and improved nutritional content.
• Opponents say resistance to Bt toxin and
  glyphosate will occur, and cite safety concerns
  about eating transformed plants.

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Figure 17.9a
Plant with crown gall disease
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Figure 17.9b
1. A. tumefaciens cells contain a Ti (Tumor
inducing) plasmid.
2. A section of DNA from the Ti plasmid, called
T-DNA, incorporates into the chromosomes of cells
infected by the bacterium.
3. When transcribed, Ti genes induce the affected
cell to begin growing and dividing. The resulting
gall protects a growing number of Agrobacterium
cells.